Entamoeba histolytica infects over 500 million people annually and is a leading cause of parasitic death in humans. The infectious cycle of E. histolytica begins with the ingestion of the cyst, a non-dividing form that is able to survive in the environment due to a protective cell wall. After ingestion, the cyst travels to the intestine, where it undergoes excystation to produce the proliferative trophozoite form, which is capable of causing disease. Some trophozoites will encyst, allowing them to be excreted and to go on to infect new hosts. Unfortunately, although the developmental cascade is essential to pathogen transmission and disease causation, research into the regulation of this process has been hampered by the lack of a good in vitro system for studying E. histolytica stage conversion. We will take advantage of established methods for inducible encystation and excystation in E. invadens, a reptilian parasite highly related to E. histolytica in which high levels of both processes can be achieved in vitro. We propose to develop a transcriptome of E. invadens during the entire life cycle (encystation and excystation) using deep sequencing of RNA transcripts (RNA-seq). This method takes advantage of next generation sequencing technologies and has previously been used to develop transcriptomes in numerous systems. RNA-seq will be performed for trophozoites, during cyst formation (8, 12, 24 and 48 h), mature cysts (72h), and early (30min, 2h, 8h) and late (48h) excysting parasites. Sequence data will be mapped to the genome and analyzed by bioinformatics methods to identify developmentally regulated genes. Information on gene annotation and alternative splicing can also be gleaned from the RNA-seq approach. Data will be deposited in EuPathDB where it will readily be available to the community. This work will provide insight into the regulation of excystation and encystation in Entamoeba and generate a wealth of avenues for future studies on the mechanisms of these vital processes. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. The parasite converts between two life-cycle stages;a stage that is important for disease transmission and a stage that causes invasive disease. We are interested in understanding the genes involved in stage conversion in this organism as these genes would be valid targets for therapeutic interventions.